1. Field of the Invention
The invention relates generally to the field of histology. In particular, the present invention is directed to an apparatus and method for automatically producing tissue slides.
2. Description of Related Art
Histology, the study of the groups of tissues found in most multicellular plants and animals, often requires that the plant or animal tissue be provided in thin sections for study. One known technique to create thin sections of tissue is through the use of a microtome. The function of the microtome is simplistic and very much like that of any mechanical slicer which carves a pre-set measured slice and retains and advances that unit of measure for each subsequent cutting cycle. The cutting results are very much dependent upon a technician""s practical skill.
Typically, tissue samples for general pathology examination are surrounded by a support medium and manually molded into standardized blocks. Paraffin is a known and commonly-used as a support medium. TissueTek O.C.T., manufactured by Sakura Finetek and available from VWR Scientific, West Chester, Pa., may also be used as a support medium. The optimum cutting temperature (O.C.T.) formulation of water-soluble glycols and resins in TissueTek O.C.T. provides a convenient specimen matrix for cryostat sectioning at temperatures of xe2x88x9210xc2x0 C. and below. Resins and polymers, such as methacrylates, may also be used as support media. Although resins and polymers are primarily used for electron microscopy, they can also be used for standard microscopy with some adjustments for hardness of the final product. Suitable resins and polymers, including Araldite 502 Kit, Eponate 12(trademark) Kit, and Glycol Methacrylate (GMA) Kit, are available from Ted Pella, Inc., Redding, Calif.
The tissue sample is manipulated in a metal support medium block mold by the technician so as to locate it close to what will become the top center surface of the support medium block. This top surface then becomes the working surface of the sample block. The sample block preparation process will be described using paraffin as the support medium.
As the tissue sample is being located and set within the support medium block mold, melted paraffin is added. The paraffin cools and hardens inside the block mold; and as it does, a plastic holder is inserted. In the hardening process, the bottom half of the paraffin block becomes locked within the frame of the plastic holder which becomes the base of the sample block. A technician then transfers the sample block to a microtome to prepare very thin sections of the tissue sample for later observation.
The prepared sample blocks are manually locked into an adjustable clamp assembly of the microtome. The technician can make limited manual adjustments to the clamp assembly in order to present the sample block""s working surface to the microtome""s cutting blade at an angle judged to be most favorable to yielding the best possible sample slice. The clamp adjustment for a given sample block is a subjective choice based on the judgement and experience of the technician.
The technician can make additional microtome adjustments that pre-set the starting lineal distance between the clamp assembly and the assembly holding the cutting blade, as well as the thickness of the sample slice each time the clamp assembly is cycled by moving it past the blade assembly. There is also limited adjustment of the angle of the cutting blade as presented to the clamp assembly; however, the optimum setting of the blade is typically about 45 degrees from vertical.
One sample slice is the product of one microtome cycle. Once a technician selects the orientation of the sample block""s working surface to the cutting blade assembly""s cutting blade, several cutting cycles are required to advance the cutting blade into the block to a working surface depth, where the slices then taken are suitable for slide application. These slices may be referred to as histologic grade slices, which are slices that a pathologist or other professional would find of acceptable quality for examination purposes. In particular, histologic grade slices are slices of a relatively uniform thickness without any significant tears, folds, wrinkles, or contamination from unrelated specimens or other artifacts. Histologic grade slices are substantially flat and smooth.
When a suitable sample depth is acquired, the technician may cycle the clamp (with the sample block locked within) one to several times to obtain a series of histologic grade slices. After each slice, a mechanism in the microtome advances the block toward the knife by the desired slice thickness, which may be set by the technician. A typical slice thickness is about 4 to 8 microns, but may vary from 1 to more than 10 microns.
The technician places the slices onto the surface of warm water, located in a pan next to the microtome. The tissue sample, still secured in its paraffin matrix, floats on the surface of the water. The warmth of the water softens the paraffin, thereby removing wrinkles or other distortions in the slice. The technician may also move a sample slice about on the surface of the water to facilitate this result.
Once the technician judges that the floating tissue/paraffin matrix sample has the desired flat form, the technician brings a receiving medium, such as a microscope slide, up under the portion of the floating sample slice that captures the full tissue specimen. Repetitive success in this, again, depends upon the skill of the technician. The slide with sample is then set aside for drying, staining and evaluation, or for various molecular or histochemical analysis.
Occasionally, such as during performance of surgical procedures, it may be necessary to get a rapid diagnosis of a pathologic process. For example, a surgeon may want to know if the margins of his resection for a malignant neoplasm are clear before closing, or an unexpected disease process may be found and require diagnosis to decide what to do next, or it may be necessary to determine if the appropriate tissue has been obtained for further workup of a disease process. This may accomplished through use of a frozen section.
Frozen sections are performed with an instrument called a cryostat. The cryostat is essentially a refrigerated box containing a microtome. The temperature inside the cryostat is about xe2x88x9220 to xe2x88x9230xc2x0 C. The piece of tissue to be studied is snap frozen in a cold liquid or cold environment (xe2x88x9220 to xe2x88x9270xc2x0 C.). TissueTek O.C.T. would be a suitable support medium for frozen sections. The freezing makes the tissue solid enough to section with a microtome. The tissue sections are cut and picked up on a glass slide. The sections are then ready for staining.
Although this process is effective, it is very time consuming and very dependent on the skill of the technician. As hospitals and laboratories seek to lower operating costs, they will seek to find more efficient ways to accomplish inefficient tasks. Also, it is desirable to reduce the variation in sample preparation that may occur between different technicians.
Therefore, a need has arisen for an improved apparatus and method for producing histological slides.
It is an object of the present invention to automatically perform the combined functions of the microtome and the technician.
It is a further object of the present invention to utilize mechanical robotic mechanisms to manipulate sample blocks and slides.
It is another object of the present invention to automatically determine the optimal working surface of the tissue sample.
A method of producing thin sections from an embedded sample is disclosed. In one embodiment, the method includes first locating a sample embedded within a support medium, which may be paraffin or a similar medium. Next, the embedded sample is oriented in such a way that its working surface is presented. This orientation may entail determining the orientation of the embedded sample with respect to the cutting blade that will produce the largest cross-sectional area. Next, a slice of the sample from said embedded sample is removed and subsequently transferred to a suitable receiving medium, which may include a microscope slide.
In another embodiment, an apparatus for applying thin sections of a tissue sample to a receiving medium is disclosed. The apparatus includes a slicing means for slicing thin sections from a tissue sample. The slicing means typically includes one or more cutting blades for slicing the tissue sample. The slicing means may include a first blade assembly that slices the tissue sample to expose the tissue sample""s working surface, and a second blade assembly for producing thin sections of the tissue sample. The apparatus further includes a plurality of transfer rollers that are sequentially arranged in tangential proximity to each other, such that a thin section on the surface of one transfer roller will be transferred to the surface of the sequentially successive transfer roller. The first sequential transfer roller of is oriented in proximity to the slicing means so that a thin section sliced from said tissue sample contacts the surface of the first sequential transfer roller. A receiving medium is disposed in tangential proximity to a final sequential transfer roller, so that a thin section on the surface of the final sequential transfer roller will be transferred to the receiving medium in a substantially smooth and flat configuration.
In another embodiment, a method for applying thin sections of a tissue sample to a receiving medium is disclosed. The method includes first slicing a thin section from a tissue sample. The slicing causes the thin section to peel from the sample and adhere to a first transfer roller. Next, the thin section is transferred from the first transfer roller to an adjacent transfer roller that is in tangential proximity with the first transfer roller. Finally, the thin section is transferred from a final transfer roller to a receiving medium that is in tangential proximity with the final transfer roller. The thin section is placed on the receiving medium in a substantially smooth and flat configuration.
In another embodiment, an apparatus for automatically producing tissue slides from a tissue sample within a sample block is disclosed. The apparatus includes a holding assembly for manipulating the sample block, a blade assembly for preparing a thin section from the sample block, and a transfer roller mechanism for transferring the thin section to a receiving medium. The apparatus further includes a controller that may track the sample block and thin section.
Other objects, features, and advantages will be apparent to persons of ordinary skill in the art in view of the following detailed description of preferred embodiments and the accompanying drawings.